Chapter-2 Review of...

Chapter-2 Review of Literature


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2.1. Plastic waste: An environmental issue

With increasing rate of urbanization in India, there is an increasing demand of plastic.

The growth of plastic industry in India is quite phenomenal having a growth rate of

17% which is higher than elsewhere in the world. This increasing demand and

abilities of plastic to fulfill these demands at cheaper price has caused an increase in

consumption of plastic in last few years. Today almost all the available plastics are

manufactured synthetically. They have much versatile qualities than naturally

occurring polymers i.e. strength, lightness, durability, resistance to degradation,

processability into wide range of shapes and strength. Increasing consumption of

plastic is having significant impact both visible and invisible on environment which

becomes more severe with no proper disposal strategies available. More than 100

million tones of plastic are produced annually worldwide and the major portion is

discarded into landfills annually. These landfills do not present a proper solution to

their disposal problem and themselves are becoming problem to municipality

worldwide as they lose capacity because of the accumulation of synthetic plastics.

Disposal of synthetic plastic is also threatening the natural terrestrial, marine

environment, soil fertility, depletion of underground water. The situation is more

acute in countries such as India where economic growth as well as urbanization is

quite rapid. In India 36.5 million tones/year of municipal solid waste is generated and

plastic waste amounting to the total 2-4 million tones/years. Mounds of plastic

polyethene bags strewn across roadsides are a familiar sight in urban India. It is

worrisome to think it would take one million years for its degradation. Recycling of

the plastic can be done as a solution but it is very tedious and expensive. The major

task is the sorting of wide variety of discarded plastic which is very time consuming.

Also, the presence of a wide variety of additives such as pigments, coatings, fillers

limits the use of the recycled material. Moreover, there are certain issues related with

the plastic industry and recycling of the plastic which need immediate attention.

These issues are related to the health and hygiene of the workers involved in the

processing trade, upgrading of processing equipment used in recycling, quality of the

effluent from the recycling plant and finally the quality of the product from recycled

plastic wastes. Given this scenario of intensive use of synthetic plastics, it is crucial


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for India to check the use of plastic. The existing policies have not been able to

provide any respite against littering and its associated problem. Therefore, there is an

urgent need to adopt policies that can help in establishing an efficient waste

management process and ensure efficient resources use in country.

In search of sustainable product development there has been a lot of research to find

different ways of using energy efficient, non-toxic, renewable sources rather than

finite sources. It has been realized that the solution to the problem of ‘polymer

garbage’ is the production and replacement of non-biodegradable by a wide range of

polymers that are degradable under varying environmental conditions.

2.2. Bioplastic

Bioplastics are the most promising alternative that can change the scenario of plastic

waste management. These are an important class of biomaterial consisting of

polyesters that are widely distributed in nature. These have physio-chemical

properties resembling that of synthetic plastic but have an advantage of being

biodegradable under different environmental conditions. There are three types of

biodegradable plastic reported:

1. Photodegradable: Photodegradable plastic, having light sensitive groups

incorporated into the backbone of the polymer as additives, can be disintegrated

by using extensive UV radiations. This makes them susceptible for further

bacterial degradation (Kalia et al., 2000a).

2. Semi-biodegradable: These are starch linked plastics where starch is

incorporated to hold together short fragments of PE. Starch in the plastic is

degraded by bacteria but bacteria are turned off by PE fragments which thereby

remain non-biodegradable (Tang and Alavi, 2011).

3. Completely biodegradable: These plastics are the most promising because of its

actual and complete degradation by bacteria e.g. PHA, PLA, aliphatic polyesters. Biodegradable polymers, such as cellophane, polylactic acid and PHAs (PHAs) have

assumed increasing importance in last few years. Out of these, PHA polymers are of

prime interest as these can be produced by bacterial fermentation using renewable


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sources. In contrast, in others either the monomer composing the polymers are derived

from the fossil sources or if produced naturally like cellulose and lactic acid further

polymerization is needed under biotechnological process. PHAs are synthesized by

bacteria as energy and food reserve material in the form of granules up to the level of

90% of total Cell Dry Weight (cdw) and can be recovered directly using various

methods.

Product applications for bioplastics largely will depend on its material properties like

its strength, life span, resistance to heat and water, the ability to be used in packaging

and its cost. However, there are certain issues which are limiting the large scale

production and commercialization of bioplastic. The improper implementation of

legislative measurements and high production cost are major issues. Policies are not

effectively implemented to enhance the use of biodegradable polymers in India e.g.

for export of products in certain countries the mandatory condition of using

Biodegradable Plastics for packaging is enforced.

Expensive production process in mainly attributed to the substrate cost and efficiency

of downstream processes depending on polymer content, productivity (PHA/unit

volume/unit time), cost of maintaining an axenic, sterile conditions and recovery

processes (Chen, 2009). The cost of PHA production is around 20 times higher than

polypropylene. Using renewable cheap sources and recombinant E. coli as PHA

producer can reduce the price comparable to other biodegradable plastics such as PLA

and aliphatic polyesters but the price still remains higher than that of petroleum based

plastic. Another major factor limiting PHAs extensive use is the brittle nature of

homopolymers. Copolymers are known to attribute more strength and flexibility of

polymers and can be produced by feeding co-monomer precursor substrates.

Exploitation of bacterial strains for their ability to use renewable sources like

biowaste as substrate to accumulate PHA copolymers is the most promising strategy.

2.3. Polyhydroxyalkanoates (PHAs)

PHAs are the polyesters of hydroxyacids hydroxylated at positions 3, 4, 5 and 6, all of

which are (R)-form chiral molecules. These are accumulated by various microbes

under nutrient (N, P, S, O and Mg) limiting and excess carbon source conditions, in


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the form of storage granules as sink for carbon and reducing equivalent (Reddy et al.,

2003; Singh et al., 2009; Koller et al., 2010; Rehm, 2010). Figure 2.1a represents the

general structural formula (Keshavarz and Roy, 2010). Depending on the number of

carbon atoms in the monomer unit, PHAs can be divided into three groups: 1) Short

chain length PHAs (SCL-PHAs), which consist of 3-5 carbon atoms (Figure 2.1 b, c).

PHB, the first PHA to be discovered in B. megaterium by Lemoigne in 1923, is the

most widely studied and best characterized PHA. It has a perfectly isotactic structure

with only the (R)-configuration; 2) Medium chain length PHAs (MCL-PHAs), which

consist of 6-14 carbon atoms, and 3) Long chain length PHAs (LCL-PHAs), which

consist of 17 and 18 carbon atoms. This division of polymers into groups is based on

the substrate specificity of PHA synthases that can only accept certain

hydroxyalkanoic acids in course of polymerization that depends on strain and culture

conditions (Keshavarz and Roy, 2010).

a.

b.

c.

Figure 2.1: Structure of PHAs: a) General formula; b) P(3HB-co-3HV) and c) P(3HB-co-4HB)

PHAs have physical properties similar to petroleum based synthetic plastics and are

degradable under both aerobic and anaerobic environment. The physical properties of

PHA granules are quite distinguished between two physical states in which PHA

occurs. In the prokaryotes, PHA occurs either as inclusion bodies or as complexes of

Ca+2 and polyphosphates in the cytoplasmic membranes. In the cytoplasm of bacteria,


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the polymer aggregates to form a spherical inclusion or granule of usually 50-500 nm

in diameter. It has a amorphous hydrophobic PHA polyester at the core forming

rubbery state and is surrounded with a layer consisting of phospholipids with attached

or embedded granule-associated proteins at the surface including the PHA synthase,

PHA depolymerases, structural, and regulatory proteins (Potter and Steinbuchel,

2005; Potter et al., 2005; Grage et al., 2009). The hydrophobic polyester core is with

water as a component that prevents crystallization by acting as a plasticizer. This is

the mobile state of PHA, that is, the form that is subject to the action of synthesizing

and degrading enzymes. During extraction of granules from the cell, the phospholipid

and protein layer is damaged or lost. After isolation, extracellular PHA granule is

often crystalline having about 50-80% of crystallinity (Tsuge, 2002; Grage et al.,

2009). The molecular weight of these compounds range from 2×105 to 3×106 Da. The

densities of crystalline and amorphous PHB are 1.26 and 1.18 g/cm3, respectively.

The Mw of PHB produced from wild-type bacteria is usually in the range of 1×103 to

3×106 g/mol. Thus, PHAs have sufficiently high molecular mass to have polymer

characteristics that are similar to conventional plastics such as polypropylene

(Madison and Huisman, 1999; Tsuge, 2002).

A few PHAs, such as PHB and copolymers of 3HB, 3HV and/or 4HB are produced

by various industries (Biocycle, Biomer, Biopol, Enmat, Mirel, and Nodax) (Chen,

2009). The history of commercialized PHAs goes back to 1959. W. R. Grace and

Company produced PHB in the U.S. for possible commercial applications. However,

the company shut down the process due to low production efficiency and a lack of

suitable purification methods. In 1970, P(3HB-co-3HV) was commercialized by

Imperial Chemical Industries Ltd. (ICI/Zeneca BioProducts, Bellingham, UK) under

the trade name of Biopol™ (Chen, 2009; Chanprateep, 2010; Meyer, 2011). In 1996,

the technology was sold to Monsanto and then to Metabolix, Inc. Now a days, a

number of different companies are developing PHAs for use in plastics worldwide,

e.g. Kaneka in Japan, and P&G Chemical, BP and Metabolix (DeMacro, 2005; Noda

et al., 2005) in the US and Imperial Chemical Industries in the UK (Khanna and

Srivastava, 2005). Kaneka and P&G Chemical have teamed up to commercialize a

product called Nodax (also known as Nodak™) which is a specialized PHA. Nodak


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™ has already been made into a variety of different prototype objects such as plastic

fiber or twine and molded plasticware such as plates and cups (Noda et al., 2005).

Metabolix is already producing preliminary PHA materials, but is teaming up with BP

for two years to produce bioplastics.

2.4. PHA biosynthesis

PHAs are synthesized by diverting intermediates of carbon metabolism to hydroxyacyl-

CoA thioesters. Pathways for PHA synthesis consist of three enzymatic reactions

catalyzed by successive action of β-Ketoacyl-CoA thiolase (phbA), acetoacetyl CoA

(phbB) reductase, PHA polymerase (phbC) (Madison and Huisman, 1999). PHA

synthase or polymerase is the key enzyme that in various pathways catalyses the

committed step of polymerizing (R)-3-hydroxyacyl-CoA thioester monomers into

polyester with the release of CoA. The nucleotide sequences of 59 PHA synthase genes

have been obtained from 45 different bacteria (Rehm 2003). The multiple alignments of

the primary structures of PHA synthases show an overall identity of 8-96% with only

eight strictly conserved amino acid residues (Rehm, 2003). Depending upon the

primary structures deduced from these sequences, the substrate specificities of the

enzymes and the subunit composition, PHA synthases differ in different organisms

(Rehm, 2006; Valappil et al., 2007a). Four major classes of PHA synthases can be

distinguished (Table 2.1). Class I and class II PHA synthases comprise enzymes

consisting of only one type of subunit (PhaC) with molecular masses between 61 kDa

and 73 kDa. Class I PHA synthases preferably utilize CoA thioesters of various (R)-3-

hydroxy fatty acids having 3 to 5 carbon atoms, whereas class II PHA synthases

preferentially utilize CoA thioester of various (R)-3-hydroxy fatty acids containing 6 to

14 carbon atoms. In class III PHA synthases two different types of subunits are present:

(i) the PhaC subunit (molecular mass of approx. 40 kDa) showing amino acid sequence

similarity of 21-8% to class I and II PHA synthases and (ii) the PhaE subunit (molecular

mass of approx 40 kDa) with no similarity to PHA synthases. These PHA synthases

prefer CoA thioesters of (R)-3-hydroxy fatty acids comprising 3 to 5 carbon atoms.

Class IV PHA synthases resemble the class III PHA synthases, but PhaE is replaced by

PhaR (molecular mass of approx. 20 kDa) which is different from the transcriptional


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regulator PhaR in R. eutropha. The PHA synthases of R. eutropha, Pseudomonas

aeruginosa, Allochromatium vinosum and B. megaterium represent Classes I, II, III, and

IV respectively (Rehm 2003; Naik et al., 2008).

Table 2.1: Four classes of PHA synthases (PhaC) (Rehm, 2006)

All PHA synthases share a conserved cysteine (Cys319 in R. eutropha PHA synthase)

as a catalytic active site to which the growing PHA chain is covalently attached. The

active-site cysteine, along with other conserved amino acids (histidine and aspartate),

constitutes a catalytic triad similar to esterases. Comparison of PHA synthase amino

acids sequences with sequences of esterases with known structures strongly suggests

that PHA synthases have an α/β-hydrolase fold.

Pathways

There are different pathways for PHA synthesis varying among different type of

organism (Suriyamongkol et al., 2007).

2.4.1. Biosynthetic pathway in R. eutropha

Most of the organisms synthesize PHA using this pathway, well established in R.

eutropha, Zoogloea ramigera, and Azotobacter beijerinckii. In R. eutropha,

metabolism of carbohydrates leads to the biosynthesis of PHA (Figure 2.2). β-

Ketothiolase, encoded by phaA, condenses two molecules of acetyl-CoA to

acetoacetyl-CoA which is subsequently reduced to (R)-3-hydroxybutyryl-CoA by the

NADPH-dependent acetoacetyl-CoA reductase, encoded by phaB. The PHB synthase,

encoded by phaC then polymerises the (R)-3-hydroxybutyryl CoA enantiomer into

the polyoxoester PHB (Tsuge, 2002; Naik et al., 2008). When propionic acid is used


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as a substrate acetyl-CoA is formed by the elimination of carbonyl carbon from

propionyl-CoA. Two moles of acetyl-CoA are used to form a HB unit of the

copolymer, while a HV unit is formed by the reaction of acetyl CoA and propionyl-

CoA resulting in final production of copolymer P(HB-co-HV) (Naik et al., 2008).

Figure 2.2: PHA biosynthetic pathway in Ralstonia eutropha (Tsuge 2002; Naik et al., 2008)

2.4.2. Biosynthetic pathway in Rhodopseudomonas rubrum

This pathway is similar to the R. eutropha pathway and found in R. rubrum where β-

oxidation of fatty acid leads to the biosynthesis of PHA (Figure 2.3).

Figure 2.3: PHA biosynthetic pathway from fatty acids oxidation in Rhodopseudomonas

rubrum (Naik et al., 2008)


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The first reaction remains the same as that for R. eutropha leading to the formation of acetoacetyl CoA, which is then reduced into L-(+)- 3-hydroxybutyryl CoA by a NADH dependent reductase. Two enoyl-CoA hydratases are also involved in the second step of catalyzing the conversion of L-3-hydroxybutyryl-CoA to D-3- hydroxybutyryl-CoA via crotonyl-CoA (Tsuge, 2002; Khanna and Srivastava, 2005; Naik et al., 2008). 2.4.3. Biosynthetic Pathway in Pseudomonas group I

This type of PHA biosynthetic pathway is found in most Pseudomonas species, belonging to rDNA homology group-I e.g. P. oleovorans. These organisms produce MCL-PHAs (from C6-C9) from MCL-alkanes, alcohols, or alkanoates using intermediates from fatty acid β-oxidation pathway (Khanna and Srivastava, 2005). SCL-PHAs, i.e., PHB homopolymer and PHB-PHV copolymer, cab be produced by these organisms in small amount i.e. less than 1.5%.

2.4.4. Biosynthetic pathway in Pseudomonas group II

This is the fourth type of PHA biosynthetic pathway found in Pseudomonas belonging to rDNA homology group-II e.g. P. aeruginosa (Figure 2.4). Most Pseudomonads from the rDNA homology group I except P. oleororans also produce MCL PHAs using this pathway. Synthesis of PHA results from de novo fatty acid synthesis pathway which involves the synthesis of copolymers of MCL 3-hydroxyalkanotae (3HA) from acetyl-CoA utilizing unrelated substrates, e.g., gluconate or acetate (Tsuge, 2002; Naik et al., 2008).

Figure 2.4: PHA biosynthetic pathway from “de novo fatty acid synthesis” in Pseudomonas

group II (Naik et al., 2008)


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2.5. Homopolymer vs Copolymer

Approximately 150 different PHA monomers have been characterized. Besides 3-, 4-,

5-, and 6-hydroxyalkanoates, different functionalized hydroxyalkanoates such as

those with halogenated and aromatic side chains have been described as constituents

of PHAs (Suriyamongkol et al., 2007). The crystalline homopolymer PHB melts in

the range of 170-180 °C and have a glass transition temperature around 4 °C. PHB in

the amorphous fraction is characterized by the same glass transition temperature as

PHB in native granules (Tg ≈ 0 °C). The crystalline behavior of the polymer (55-80%

crystallanity) results in fairly stiff and brittle materials, somewhat limiting its

applications and processability (e.g. elongation to break is about 2-10% compared to

up to 400% for some polyolefins). The tensile strength of PHB (43 MPa) is close to

that of polypropylene (38 MPa) (Potter and Steinbuchel, 2005; Albuquerque et al.,

2011). It is well established that PHA’s thermal and mechanical properties depend

directly on the polymer composition and structure. In contrast to homopolymer PHB,

copolymers are more ductile, tensile, easier to mold with increased flexibility (higher

elongation to break), having more favorable thermoplastic properties, better film

forming capacity and mechanical properties similar to low-density polyetylene. These

features improve their strength and processability (Tsuge, 2002; Valappil et al.,

2007b; Lee et al., 2008; Albuquerque et al., 2011). The incorporation of different

monomer types reduces polymer crystallinity by disturbing the crystal lattice. There

are studies reporting that melting and glass transition temperature of P(3HB-co-3HV)

steadily decrease from 0 to 30 mol% fraction of 3HV (Lee et al., 2008; Allen et al.,

2010; Bengtsson et al., 2010a). The lower melting temperatures of these copolymers

allow processability for a wider temperature range. Table 2.2 summarizes the physical

properties of commonly used PHAs and synthetic polymers.

Copolymer production can be achieved by feeding copolymer precursors substrates

where their composition can be varied with different feeding strategies (Valappil et

al., 2007b; Singh and Mallick, 2008). The use of continuous feeding was observed to

increase the HV content relatively in comparison to pulse wise feeding (Albuquerque

et al., 2011; Hafuka et al., 2011). There have been reports on the production of the

terpolymer P(3HB-co-3HV-co-4HB) reported for the first time by Chanprateep and


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Kulpreecha by the newly isolated Cupriavidus necator strain A-04 (Chanprateep and

Kulpreecha, 2006). Here, the terpolymer with the highest (93 mol%) 4HB mole

fraction units P(4% 3HB-co-3% 3HV-co-93% 4HB) was produced. This terpolymer

showed an elongation of 430%, a toughness of 33MPa, and a Young's modulus of

127MPa, similar to those of low-density polyethylene whereas the terpolymer P(11%

3HB-co-34% 3HV-co-55% 4HB) showed a Young's Modulus of 618MPa, similar to

that of polypropylene. Biodegradable plastic, marketed under the trade name

"BIOPOL" was produced industrially for first time by ICI Ltd. in 1982, has 20 mol%

of 3HV content.

Table 2.2: Comparison of the physical properties of specific PHAs and commonly used

synthetic polymers (Castilho et al., 2009; Akaraonye et al., 2010)

Polymer Melting temperature

(°C)

Glass transition

temperature (°C)

Young’s modulus

(GPa)

Elongation to break

(%)

Tensile strength (MPa)

PHB 175-180 4 3.5-4 3-8 40

P(4HB) 53 -48 149 1000 104

P(3HB-co-20% 3HV) 145 -1 1.2 50-100 20-32

P(3HB-co-10% 4HV) 159 - - 242 24

P(3HB-co-16% 4HB) 150 -7 - 444 26

P(3HB-co-10% 3HHx) 127 -1 - 400 21

P(3HB-co-6% 3HD) 130 -8 - 680 17

High-density polyethylene 112-132 - 0.4-1.0 12-700 17.9-33.1

Low-density polyethylene 88-100 - 0.05-0.1 150-600 15.2-78.6

Polypropylene 170-176 -10 1.0-1.7 400-900 29.3-38.6

Polystyrene 110-240 100 3.0-3.1 3-4 50

Nylon-6,6 265 - 2.8 60 83

2.6. Biowaste as cheap and renewable substrate

Although the PHAs are commercial for last 20 years but production of biodegradable

plastic on large scale at comparable cost is the main focus in this area. BIOPOL costs

around $16/kg which is much higher than the synthetic plastic which costs $1/kg. This

high cost is mainly because of the expenses of carbonaceous substrate which


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contribute around 45% of the total production cost (Reddy et al., 2003; Kumar et al.,

2009). Use of renewable sources like biowaste as cheap carbon source can reduce the

cost attributed to substrate as well as can also solve another problem related to the

handling and treatment of large amount of waste.

2.6.1. Waste management

Waste management is becoming political priority in many developed and developing

countries. Waste management methods used today commercially include: i)

transportation of waste to low lying areas/landfills, ii) burning of waste on site or in

the incinerators, iii) composting, iv) briquetting, (v) recycling of waste matter and vi)

microbial treatment (aerobic and anaerobic) etc. As each of these methods has its own

advantages and can be employed to certain types of waste an integrated approach is

required for complete disposal of waste (Talyan et al., 2007). Disposal of wastes in

unorganized landfills causes environmental pollution due to slow and uncontrolled

fermentation, which generates gases like CO2, CH4 and H2S. Natural degradation in

landfills is very slow continued over scores of years and only 10-15% of the available

energy content can be recovered. Burning of wastes on site is a conventional method

of disposing wastes. Out of the 4000-5000 tons of waste generated per day in

metropolitan cities about 60-70% is collected and discarded into landfills. Burning is

possible only for dry wastes and these wastes have up to 65% of materials, which can

be combusted. However, it results in generation heat at the rate of around 2700

BTU/kg waste (1 BTU, British thermal unit = 2.9x 10-4 KWH) (Kalia, 1991) and

obnoxious gases affects the environment and human health. Composting is the highest

form of recycling. Composting is inexpensive, rapidly implemented and a publicly

acceptable treatment process that recycle the organic matter to be reused in beneficial

manner. Briquetting process consists of heating the feedstock’s under controlled

conditions, without any contact with air and converts highly voluminous and

troublesome waste materials in to clean, non-polluting fuel. Being compact,

transportation and storage costs are reduced. It is environmentally superior technology

for handling different ligno-cellulosic materials (Kalia, 1992, 2007, Kalia et al.,

1992a, b; 1995; McDowall and Eames, 2007).


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2.6.2. Anaerobic digestion

The fermentation technology is the best technology for treatment and recycling of

organic wastes being more hygienic and convenient process than other means like

briquetting, burning, composting and landfilling. Anaerobic digestion (AD) process is

one of the oldest processes used for the treatment stability of waste materials. AD is a

series of processes in which microorganisms break down biodegradable material in

the absence of O2. Unlike uncontrolled and slow fermentation of wastes in a landfill,

biodegradation is using AD system results in energy generation and production of

nutrient rich biomanure providing a better approach for waste recycling and

stabilisation. The intermediate of this process are primarily converted to H2 and

finally in to CH4 and CO2 during methanogenesis (Kalia and Luthra, 1994; Kalia,

1995; Kumar et al., 1995; Kalia, 2007; Chen et al., 2008; Holm-Nielsen et al., 2009).

2.6.3. Steps involved in anaerobic digestion

There are four key stages of AD process:

(i) Hydrolysis

Through this first step of AD the complex organic molecules are broken down into

simple sugars, amino acids and fatty acids. Extracellular enzymes such as such as

amylase, cellulase, lipase and protease, which are excreted by hydrolytic fermentative

bacteria catalyse this hydrolytic process which provide substrates to next group of

bacteria (Kalia et al., 2000a, b).

(ii) Acidogenesis

The biological process of acidogenesis leads to further breakdown of the remaining

components by acidogenic (fermentative) bacteria. This step is fastest in the AD

processes with acidogenic bacteria having with minimum doubling time of around 30

min. Here, low molecular weight compounds such VFAs are created along with NH3,

CO2 and H2 well as other byproducts.


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(iii) Acetogenesis

In the third stage AD simple molecules created through the acidogenesis phase are

further digested by acetogens to produce largely acetic acid as well as CO2 and H2.

The acetogens are slow growing, doubling time of around 1.4 days and among them

more co-operations are needed for efficiency.

(iv) Methanogenesis

Biological process of methanogenesis constitutes the last step where methanogens

catabolize acetate and a mixture of H2 + CO2 to CH4. Methanogens are unique among

prokaryotes and are classified as members of archaebacteria, a group of

phylogenetically different organisms.

All these processes occur simultaneously and synergistically, the first group has to

perform its metabolic action before the next stage bacterial group take over and so

forth. AD of animal manure offers several environmental, agricultural and socio-

economic benefits throughout production of biogas as clean and renewable fuels

(Holm-Nielsen et al., 2009; Lew et al., 2009). Biological oxygen demand (BOD) and

chemical oxygen demand (COD) are reduced by these pathways.

Figure 2.5: Strategy for efficient degradation of biological wastes (Kalia and Purohit, 2008)


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2.6.4. Anaerobic Digestion and PHA production

AD is generally accepted to be effective and economical method for treatment of

waste treatment in which pollution control and energy recovery can be achieved. At

present anaerobic treatment is successfully implemented for various types of

industrial as well as domestic wastes. Hydrolyzing bacteria solubilize and convert

complex organic waste into simpler and utilizable products (VFAs and other soluble

organic compounds) in acidogenic stage og anaerobic digestion process (Figure 2.5).

These hydrolysis products can be utilized by microbial culture as substrate for

bioproduct formation such as PHA (Sonakya et al., 2001; Sun et al., 2007). Various

agricultural, industrial and food waste are being exploited for this purpose e.g.

extruded rice bran, municipal sludge, palm oil, paper mill waste water, soy molasses,

sugar cane molasses, waste cooking oil, and whey, etc. (Khardenavis et al., 2007;

Bengtsson et al., 2008; Castilho et al., 2009; Ntaikou et al., 2009; Chanprateep,

2010). So, we need to search and exploit microbial diversity for bacteria having

ability to produce PHA in high concentration as well as utilize inexpensive sources as

substrate (Kalia et al., 2003; Kalia and Purohit, 2008). The best PHAs-producing

species should satisfy several demands such as the fast growing population, being

able to utilize cheap carbon and having a high production rate.

2.6.5. Potential substrates for PHA production

To make PHAs production more economical, some researchers have tried to produce

PHAs from inexpensive carbon sources. Among various substrates used for PHA

production starch, methanol, dairy byproducts, agro-industrial byproducts, oily waste

are generally used (Tian et al., 2009; Akaraonye et al., 2010). These are readily

utilized by various bacterial strains belonging to gram negative and gram positive

(Table 2.3 & 2.4) group as potential substrates for economic PHA production.

(i) Starch

Starch is a potential carbon feedstock that can be used for PHA production as it is

quite cheap and is readily available in large amount from plant sources (Chen et al.,

2006; Huang et al., 2006). However, to utilize starch as carbon source in PHA

production it needs to be hydrolyzed first because it is complex in nature (Huang et


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al., 2006; Halami, 2008). P(3HB-co-3HV) production has been reported from

Haloferax mediterranei with enzymatically extruded starch in pH stat fed batch

fermentation process achieving total 1.14 g/L biomass with PHA content of 0.84 g

amounting to 43% of total cdw. Here, the solution feed contained extruded starch/

yeast extract in ratio 1/1.7 g/g (Chen et al., 2006). Halomonas boliviensis produced

1.2 g/L biomass containing 56% of cdw PHB in shake flask cultures when starch

hydrolysate was used. However, under batch fermentation, the PHB content was 35%

of cdw under optimum O2 supply and increased to 41% of cdw under O2 limitation

during active growth but biomass was adversely affected (Quillaguaman et al., 2005).

PHA production has also been reported using B. cereus CFR06 which under batch

culture produced 48% of cdw PHA and 1 g/L biomass (Halami, 2008).

Saccharification hydrolyse the starch into more utilizable sugars that can further be

utilized for PHA production. Saccharified waste potato starch was used as carbon

source where 94 g/L PHB was produced. It was equivalent to 55% of total cdw in fed

batch fermentation with R. eutropha NCIM 5149 (Haas et al., 2008).

(ii) Methanol

Methanol produced by anaerobic digestion of woody material and organic substrates

can be used as cheap carbon source for PHA production. Species belonging to

Methylobacterium are shown to utilize methanol for PHA production.

Methylobacterium sp. GW2 has been reported to produce maximum PHB content (out

of cdw) of 40% utilizing methanol as substrate. The PHB accumulation in this

organism is growth associated thereby removing the need for two-stage fermentation

(Yezza et al., 2006). Homopolymer PHB production was reported from methanol

using a 2 L fed-batch fermentation using Methylobacterium extorquens. Here, a total

biomass of 9 g/L and PHB content (out of cdw) of 30-33% were achieved. However,

the organism was able to accumulate copolymer of P(3HB-co-20-mol% 3HV) when

valerate was added with methanol (Bourque et al., 1992). Mokhtari-Hosseini et al.

carried out PHB production in 5L fermentor achieving total biomass of 15.4 g/L with

PHB content of 62.3% on methanol as substrate using M. extorquens DSMZ 1340

(Mokhtari-Hosseini et al., 2009). Recently, mixed culture has been shown to utilize

methanol as substrate under a non-sterile process and producing PHB to the level of


22

0.424 g/L while the biomass was achieved to level 1.54 g/L. Here, optimum PHB was

produced with methanol concentration of 0.15% (v/v) (Dong et al., 2011).

(iii) Agro-industrial waste

A number of agricultural wastes like wheat bran, rice bran, corn starch, molasses, PS and

other agricultural byproducts are shown to be potential substrates for PHA production.

H. boliviensis LC1 was shown to produce 1.08 g/L of PHA equivalent to 33.8% of cdw

on wheat bran hydrolysate in a shaken flask study (Van-Thuoc et al., 2008). Huang et al.

have successfully used inexpensive extruded rice bran and corn starch in PHA

production from H. mediterranei (Huang et al., 2006). Here, PHA concentration and

PHA content of 77.8 g/L and 55.6% of cdw, respectively, were achieved using extruded

rice and extruded corn starch in the ratio of 1: 8 in a pH-stat fed-batch fermentation.

Molasses contain large amount of sugars, sucrose being predominant (appox. 50%) and

therefore can be considered as a potential source for PHA production (Solaiman et al.,

2006; Akaraonye et al., 2011). Mixed bacterial culture have used for PHA production on

sugar cane molasses in a sequencing batch reactor (SBR) operated under aerobic

dynamic feeding (Albuquerque et al., 2010a, b; Bengtsson et al., 2010a, b). Albuquerque

et al. achieved the production of 30% of cdw P(3HB-co-3HV) with a biomass of 3.5 g/L.

Bacterial species belonging to Bacillus were also shown to utilize molasses for PHA

production (Santimano et al., 2009). Further, Solaiman et al. successfully utilized soy

molasses for the production of MCL-PHA P(3HDD-co-3HO-co-3HTD) using P.

corrugate. A cell density of 3.4 g/L and a total PHA content of 5-17% cdw were

achieved when 5% (w/v) soy molasses was added to the E-medium (Solaiman et al.,

2006). Recently, PS were also shown in our laboratory as potential substrate for PHB

production where biowaste slurry was successfully supplemented upto 50% of total feed

(Kumar et al., 2009). Further improvement in using only hydrolyzed PSS as medium

was achieved for successful PHA production (Patel et al., 2011b). Conversion of agro-

industrial waste water into PHB was successfully employed by various researchers

(Khardenavis et al., 2007). Rice grain based spent wash has produced PHB yield upto

67% of cdw while the yield was upto 42.3% with jowar grain based distillery spentwash

(Khardenavis et al., 2007). Mixed cultures enriched under feast and famine conditions

have been used produce PHA from VFAs obtained in fermented paper mill wastewater.


23

Here the yield achieved was 0.11 kg PHA/kg COD treated containing 53.69 mol% of

3HV (Bengtsson et al., 2008).

(iv) Dairy byproducts

Whey has been the most extensively for PHA production by various micro-organism.

It represents about 80-90% of the volume of processed milk. Only about half of the

whey produced is converted into useful products such as human and animal feed

while the rest is disposed of as waste causing environmental problems due to its high

oxygen demand. Koller and co-workers have found that hydrolyzed whey can be used

by P. hydrogenovora to accumulate up to 5 g/L biomass containing 1.27 g/L and

12% of cdw PHB monomers (Koller et al., 2008). Recombinant E. coli has been used

extensively for the purpose and a PHB yield of 168 g/L contributing to 87% of cdw

was achieved on whey in feed containing 280 g lactose/L under fed batch culture

(Ahn et al., 2001). Using E. coli K24K, Nikel and collegues achieved 72.9% of cdw

PHB amounting to 51.1 g/L under pH controlled fed-batch fermentation with whey

and corn steep liquor as carbon and nitrogen sources was used PHA production

achieving (Nikel et al., 2006 ). Thermus thermophilus utilized whey as substrate to

produce PHA upto the content of 35% containing 3HV), 3HHp, 3HN and 3HU on

media containing 24% (v/v) whey under nutrient limitation (Pantazaki et al., 2009).

Koller et al., have reported successful PHA production using Hydrogenophaga

pseudoflava on whey using an antibiotic (vancomycin) based strategy against the B.

cereus contamination (Koller et al., 2011b). Thus, the successful use of whey as a

carbon source for PHA production is certainly a step forward towards the reduction of

the cost of PHA production.

(v) Oily waste

Improper disposal of the waste vegetable oil leads to an increase in the biological

oxygen demand (BOD) and chemical oxygen demand (COD), which in turn leads to the

deoxygenation of water, an infiltration into soil sediments and aquifer contamination.

These waste vegetable oils can be utilized as inexpensive carbon sources for PHA

production (Mumtaz et al., 2010). This will not only help in converting waste to a

useful material but also aid in the disposal management of the waste vegetable oil.


24

Various researchers have shown PHA production using oily wastes like corn oil, olive

oil, olive oil waste water (alpechin), palm kernel oil and soybean oil. There are various

reports of PHA production from C. necator on PKO (Sudesh et al., 2011). Production

of P(3HB-co-3HV) upto 79% of cdw by C. necator was reported when grown on palm

kernel oil with sodium valerate and propionate as 3HV precursors which ranged from 0-

60 mol% (Bhubalan et al., 2008). Copolymer, P(3HO-co-3HD-co-3HDD) upto 63% of

cdw was achieved using P. guezenneibiovar tikehau when coprah oil was used as the

main carbon substrate (Simon-Coin et al., 2008). Different copolymer (P(3HB-co-

3HHx) with different %mol ratios of 3HHx production has been reported using

recombinant strains harbouring the genes mostly belonging to Aeromonas and

Pseudomonas. P. putida KT2442 strain KTT2 on olive oil waste water (alpechin)

produces a maximum PHA concentration and PHA content of 0.1 g/L and 3.59% of

cdw respectively with a corresponding biomass accumulation of 4.2 g/L (Ribera et al.,

2001). PHA production by Pseudomonas sp. on rapeseed oil (Mozejko et al., 2011) and

P. oleovorans on jutropha carcus oil (Allen et al., 2010) have been reported recently. In

a recent report, C. necator produced PHB on derivatives of rapeseed oil upto 1.2 g/L

with waste frying oil which was higher than that observed with pure oil (0.62 g/L) and

heated oil (0.9 g/L) (Verlinden et al., 2011).

2.7. PHA producing microorganisms

PHAs play an important function in ecosphere. PHA producing microorganisms

represents a widespread phylogenetic diversity and are present in ecosystems where

fluctuating availability of C source and nutriens prevails (Koller et al., 2011a).

Although more than 300 different microorganisms belonging to 90 genera are known

so far to synthesize PHAs (Hazer and Steinbuchel, 2007), a perusal of the capacities

of different microbes to produce homopolymers and copolymers of PHAs reveals that

Alcaligenes, Pseudomonas and Ralstonia belonging to Gram-negative lead this group.

These have abilities to utilize pure substrates, agricultural wastes, oily wastes, dairy

products and CO2 for PHA production at large scale (Singh et al., 2009) (Table 2.3 &

2.4). Recombinant strain of E. coli harboring genes for PHA synthesis have also been

used extensively (Suriyamongkol et al., 2007). Among gram positive group Bacillus

sp. are most commonly used having the potential to utilize wide range of substrates

for PHA production (Table 2.3 & 2.4).


25

Table 2.3: Microbial biodiversity of PHA homopolymer producers

Substrate Organism Polyhydroxybutyrate Reference g/L % of cdw

Gram negative group

Glucose

Aeromonasa 0.49 36.8 Chien and Ho, 2008 Azotobacter 3.7 77.3 Myshkina et al., 2008 Comamonas 1.27 53 Lee et al., 2004 Escherichiaa 51.1 72.9 Nikel et al., 2006 Pseudomonas 0.06 5.7 Bertrand et al., 1990 Ralstonia 0.97 84 Nurbas and Kutsal, 2004 Vibrio 0.02 14.2 Chien et al., 2007

Fructose Comamonas 0.05 3.0 Lee et al., 2004 Ralstonia 5.5 19.9 Khanna and Srivastava, 2008

Sucrose Alcaligenes 5.48 67.4 El-sayed et al., 2009 Comamonas 0.04 2.0 Lee et al., 2004 Vibrio 0.17 45.5 Chien et al., 2007

Lactose

Comamonas 0.07 4.0 Lee et al., 2004 Hydrogenophaga 0.05 - Povolo and Casella, 2003 Methylobacterium 1.16 - Nath et al., 2008 Paracoccus 0.03 - Povolo and Casella, 2003 Pseudomonas 0.15b 56.0 Young et al., 1994 Sinorhizobium 0.01 14.0 Povolo and Casella, 2003

Fatty acids (& derivatives)

Brachymonas - 64.0 Shi et al., 2007 Comamonas 0.08 4.0 Lee et al., 2004 Escherichia 1.20 54.3 Chien et al., 2010 Pseudomonas 70.0-80.0 Lopez et al., 2009 Spirulina - 10.0 Jau et al., 2005 Vibrio 0.07 12.1 Chien et al., 2007

Maltose Comamonas 0.02 1.0 Lee et al., 2004 Methanol Methylobacterium 15.4 62.3 Mokhtari-Hosseini et al., 2009

Starch

Aeromonas a 0.60 32.7 Chien and Ho, 2008 Azotobacter 3.2 63.0 Myshkina et al., 2008

Haloferax 6.48 32.4 Lillo and Rodriguez-Valera, 1990

Protomonas - - Suzuki et al., 1986

Glycerol

Escherichia a (batch) 3.52 42.0 Nikel et al., 2008 (fed batch) 10.81 51.0 Methylobacterium 10.5 50.0 Bormann and Roth, 1999 Ralstonia - 70.0 Mothes et al., 2007 Vibrio 0.19 42.8 Chien et al., 2007

Xylose Burkholderia 36.0 60.0 Silva et al., 2004 Pseudomonas 0.41 19.7 Bertrand et al., 1990


26

Substrate Organism Polyhydroxybutyrate Reference g/L % of cdw

Agricultural Waste

Alcaligenes - - Wang et al., 2007 Azotobacter 22.0 66.0 Page and Cornish, 1993 Burkholderia 2.73 62.0 Silva et al., 2004 Escherichia a 31.6 80.0 Liu et al., 1998 Haloferax 23.0 27.0 Huang et al., 2006 Klebsiella a - 70.0 Zhang et al., 1994 Pseudomonas 0.94 57.7 Aremu et al., 2010 Ralstonia 94.0 52.5 Haas et al., 2008

Dairy Products

Escherichia a 51.1 72.9 Nikel et al., 2006 Hydrogenophaga 0.02 4.4 Povolo and Casella, 2003 Methylobacterium 3.91 66.0 Nath et al., 2008 Pseudomonas 1.27 12.0 Koller et al., 2008 Sinorhizobium 0.02 3.5 Povolo and Casella, 2003

Oily Waste Ralstonia 0.84b 79.0 Budde et al., 2011

Industrial Waste Azotobacter 2.4 48.2 Sathesh Prabu and

Murugesan, 2010 Burkholderia 2.47 57.4 Alias and Tan, 2005 Pseudomonas 22.0 70.0 Jiang et al., 2008

Gram positive group

Glucose Bacillus

0.43 50.0 Kumar et al., 2009; 0.81 38.0 Valappil et al., 2007b

Streptococcus - - Yuksekdag et al., 2008 Streptomyces 1.5-11.8 Verma et al., 2002

Fructose Bacillus 0.39 0.75 Kumar et al., 2009

Sucrose Bacillus

1.2 48.0 Anil-Kumar et al., 2007; 0.17 35.0 Kumar et al., 2009

Streptococcus - - Yuksekdag et al., 2008

Lactose Lactobacillus - - Yuksekdag et al., 2008 Lactococcus - - Yuksekdag et al., 2008 Streptococcus - - Yuksekdag et al., 2008

Fatty acids (& derivatives) Bacillus 0.45 80.1 Valappil et al., 2007b

Agricultural waste

Bacillus - - Akaraonye et al., 2011

3.3 41.0 Kumar et al., 2009 Staphylococcus - - Wang et al., 2007

Industrial waste

Actinobacillus 2.25 46.5 Son et al., 1996 Bacillus 8.2 51.2 Vijayendra et al., 2007

a: Recombinant strains b: g/g substrate consumed


27

Table 2.4: Microbial biodiversity of PHA copolymer producers

Substrate Organism Polyhydroxyalkanoates Reference

g/L % of cdw

Composition

Gram negative group

Glucose Pseudomonas 0.09 25.5 3HB:3HV:3HHD:3HOD Singh and Mallick, 2009

Sucrose Rhizobium 1.62 58 3HB:3HV Anil-Kumar et al., 2007 Sphingomonas 0.99 45 3HB:3HV Anil-Kumar et al., 2007

Fatty acids (& derivatives)

Aeromonasa 0.92 59.4 3HB:3HHx Chien and Ho, 2008 Azotobacter 3.41 75.8 3HB:3HV Myshkina et al., 2010 Comamonas 0.63 25.0 3HB:4HB Lee et al., 2004

Escherichiaa 0.39 44.0 3HB:3HV:3HHx: 3HO:3HD:3HDD Davis et al., 2008

Pseudomonas 0.57 38.0 3HHx:3HO:3HUde :3HNe:3HHpe Hartmann et al., 2006

Ralstonia 0.13-0.56 <84 3HB:3HV Nurbas and Kutsal, 2004

Methanol Methylobacterium 2.98 33.0 3HB:3HV Bourque et al., 1992

Agricultural waste

Haloferax 77.8 55.6 3HB:3HV Huang et al., 2006 Klebsiella - - 3HB:3HV Wang et al., 2007

Pseudomonas 0.22-0.25

7.0-17.0

3HHx:3HO:3HD: 3HDD:3HDde: 3HTD:3HTde

Solaiman et al., 2006

Rhizobium 0.71 31.0 3HB:3HV Anil-Kumar et al., 2007 Sphingomonas 0.44 22.0 3HB:3HV Anil-Kumar et al., 2007

Dairy products

Haloferax 14.7 87.5 3HB:3HV:4HB Koller et al., 2007 Pseudomonas 1.44 12.0 3HB:3HV Koller et al., 2008 Ralstonia 0.98 20.6 3HB:3HV Marangoni et al., 2002

Oily waste

Comamonas - 87.5 3HHx:3HO:3HD: 3HDD:3HTDa

Thakor et al., 2005

Pseudomonas - 26.06 3HB:3HV Allen et al., 2010

Ralstoniaa 20.0 95.0 3HB:4HB Park and Kim, 2011

Industrial waste Azotobacter 5.48 58.0 3HB:3HV Ryu et al., 2008

Gram positive group

Glucose Bacillus 0.28 32.9 3HB:3HHx Tajima et al., 2003 Microlunatus 1.41 26.0 3HB:3HV Akar et al., 2006

Fructose Bacillus 0.50 40.3 3HB:4HB Valappil et al., 2007b Sucrose Bacillus 0.64 38.4 3HB:4HB Valappil et al., 2007b

Fatty acids Bacillus 0.23 47.4 3HB:3HV Valappil et al., 2007b 0.33 64.5 3HB:3HV:4HB:3HHx Tajima et al., 2003

Agricultural waste Bacillus 2.0 54.0 3HB:3HV Anil-Kumar et al., 2007

Industrial waste Rhodococcus 0.03 2.4 3HB:3HV Fuchtenbusch and

Steinbuchel, 1999

a: Recombinant strains


28

2.7.1. Alcaligens and Ralstonia

Ralstonia and Alcaligenes are versatile organisms with well established abilities to

utilize pure substrates, agricultural wastes, oily wastes, and dairy products for PHA

production. R. eutropha which is most commonly and extensively studied for PHA

production accumulates homopolymers (PHB) to tercoploymers (P(3HB-co-3HV-co-

3HHx) from carbon sources like sugars, organic acids, vegetable oils and CO2 (Tsuge,

2002). There are several other taxonomic names of R. eutropha are found in literature

including Hydrogenomonas eutropha, A. eutrophus, Wautersia eutropha, and C.

necator (Vandamme and Coenye, 2004). Ralstonia has been shown to produce 232

g/L of PHA (82% of cdw) from glucose (Ryu et al., 1997) and up to 96 g/L of PHA

(76% of cdw) from oily wastes (Kahar et al., 2004). Terpolymers P(3HB-co-3HB-co-

3HHx) was reported from mixtures of palm kernel oil and 3HV precursors (Bhubalan

et al., 2008; Bhubalan et al., 2010). Nutritional supplementation with oleic acid

resulted in the improving the physical characteristics of PHB (Grigull et al., 2008).

PHA production by R. eutropha has been exploited in high cell density cultivation

reactor for PHB production on fructose achieving 27.7 g/L biomass with PHB

concentration of 5.5 g/L and productivity of 0.55 g/L/h (Khanna and Srivastava,

2008). Production of P(3HB-co-4HB) was reported by on sugarcane, brown

sugarcane, rock sugar, toddy palm sugar and coconut palm sugar in fed batch cultures.

Here, the mole fraction of 4HB units could be varied from 0 to 94mol% by switching

of the ratios of carbon to nitrogen, together with the ratios of fructose to c-

Hydroxybutyric acid or 1,4-butanediol. The total P(3HB-co-4HB) content was 71% of

cdw with a 4HB unit mole fraction of 30 mol% in the copolymer. The study of

kinetics of P(3HB-co-4HB) production by C. necator strain A-04 has shown that

synthesis of 4HB units was growth-associated under nitrogen-sufficient conditions

and the synthesis of 3HB units was enhanced under nitrogen deficient conditions

(Chanprateep et al., 2008). In further study, upto 112 g and 73 g P(3HB-co-4HB)

were achieved containing 38% mole fraction of 4HB content under optimum

condition (Chanprateep et al., 2010). C. necator was recently reported to produce

PHB on various derivatives of rapeseed oil (Verlinden et al., 2011). By co-feeding

soybean oil and γ-butyrolactone as carbon sources, P(3HB-co-4HB) could be


29

produced by R. eutropha KCTC 2662 with cdw of 10-21 g/L, yields of 0.45-0.56 g-

PHA/g-soybean oil used and 4HB fractions of 6-10 mol% (Park and Kim, 2011).

2.7.2. Pseudomonas

Pseudomonas has been widely observed to synthesize MCL-PHA on various aliphatic

alkenes or fatty acids, agricultural and oily wastes (Khardenavis et al., 2007; Jiang et

al., 2008). P. aeruginosa, P. oleovorans, P. resinovorans and other Pseudomonas sp.

have been reported to simultaneously produce C4 to C12 3HA units i.e. SCL-MCL-

PHAs (Singh and Mallick, 2009). Pseudomonas corrugata 388 accumulated MCL-

PHA 5 to 17% of cdw, constituted by 3HO, 3HDD, 3HDde and 3HTde on 2 to 5%

w/v soy molasses (Solaiman et al., 2006). Attempts to utilize agro-industrial oily

wastes like waste frying oil and waste free fatty acid from soybean oil as feed resulted

in 29 to 66% PHA by P. aeruginosa (Fernandez et al., 2005). P. oleovorans can

produce a natural-synthetic hybrid block copolymer polyhydroxyoctanoate-diethylene

glycol which has lesser molecular mass (50%) and reduced polydispersity. It had

similar thermal and crystalline properties as homopolymers (Foster et al., 2005).

Here, polyethylene glycol (PEG106) in 1000:1 ratio affected the polymer yield and

processing.

Recently, homopolymer poly-3-hydroxydecanoate (PHD) and P(3HD-co-84mol%

3HDD) has been reported on decanoic acid or dodecanoic acid by mutant P. puitda

KTQQ20 (Liu et al., 2011). This mutant strain was constructed by knocking out the

genes fatty acid degradation enzymes fadB, fadA, fadB2x, fadAx, 3-hydroxyacyl-

CoA dehydrogenase and acyl-CoA dehydrogenase encoded by the genes PP2136,

PP2137, PP2214, PP2215, PP2047 and PP2048 and also 3-hydroxyacyl-CoA-

acylcarrier protein transferase encoded by PhaG leading to a significant reduction of

fatty acid β-oxidation activity.

2.7.3. Haloferax

Presence of PHA in members of the family Halobacteriaceae of the archaea has also

been shown (Quillaguaman et al., 2010). Although not widely studied, certain

archaeal strains of Haloferax, Halobacterium, Haloarcula and Haloquadratum have


30

been reported for their abilities to synthesize PHA from inexpensive carbon sources as

feed material (Han et al., 2007; Huang et al., 2006). phaEC encoding PHA synthase

in H. mediterranei has been reported in various studies to produce P(3HB-co-3HV)

copolymer upto 60% of cdw from starch, glucose, or other cheaper carbon sources,

including industrial by-products (Lillo and Rodriguez-Valera, 1990; Koller et al.,

2007; Lu et al., 2008) and P(3HB-co-11% 3HV) on glucose with PHA content of

48.6% and productivity 0.36 g/L/h (Don et al., 2006). H. mediterranei has high

growth rate, metabolic versatility and genetic stability (Lu et al., 2008). Since H.

mediterranei survives in extreme salinity, it circumvents contamination problem and

consequently greatly reduces the sterility requirement. A study with the same

microorganism on hydrolyzed whey as substrate resulted in P(3HB-co-6% 3HV)

production with yield of 72% and productivity of 0.09g/L/h but P(3HB-co-21.8%

3HV-co-5.1% 4HB) was obtained with yield increased to 87.5% when sodium

valerate and γ-butyrolactone was supplemented into feed (Koller et al., 2007).

2.7.4. Recombinant E. coli

The PHA production by recombinant E. coli on different substrates has been widely

reported (Li et al., 2007b). PHA composition can be altered by engineering desirable

genes into E. coli (Li et al., 2010; Tomizawa et al., 2011). In a cell-recycle fed batch

fermentation strategy, recombinant E. coli CGSC4401 (harboring Alcaligenes latus

PHA genes) yielded 4.6 g PHB/L/h from whey (Ahn et al., 2001). E. coli

phosphotransferase system mutant strain LR1010 harboring phaCRe and phaABRe

genes from R. eutropha could simultaneously consume glucose and xylose to

accumulate SCL-PHA. Here total cdw was achieved 2.3 g/L, with PHA equivalent to

11.5% of cdw. However, E. coli LR1110, harboring phaC1 gene from P. aeruginosa

could accumulate MCL-PHA (2.3 g/L of cdw) by using a mixture of glucose and fatty

acids (Li et al., 2007a). More recent attempts of co-expression of phaA and phaB

genes from Bacillus sp. and phaC1 and phaJ1 from Pseudomonas sp. in E. coli

JC7623 resulted in production of PHA with SCL and MCL in the range of 78:22 to

18:82. The PHA yields by E. coli JC7623ABC1J1 were further improved from 28 to

34% of cdw by an additional 3 to 11% as a result of the action of acrylic acid, an

inhibitor of β-oxidation (Davis et al., 2008). Ultra high molecular weight PHB was


31

produced by recombinant E. coli (phaCABRe of R. eutropha) (Park et al., 2005)

resulting in successful preparation of PHB stretched films and improvement in

mechanical properties (Tsuge, 2002). Recently, a Novel lactic acid (LA)-based

terpolymer, P(LA-co-3HB-co-3HV), was produced in LA-overproducing mutant E.

coli JW0885 on glucose and propionate. Here, the mutant strain harbored the genes

encoding LA-polymerizing enzyme (mutant of phaC1 from Pseudomonas sp. 61-3),

phaA and phaB from R. eutropha and propionyl-CoA transferase gene from

Megasphaera elsdenii (Shozui et al., 2010). Poly (3-hydroxypropionate) (3HHp) was

recently reported with recombinant E. coli harbouring the genes for glycerol

dehydratase of Clostridium butyricum, propionaldehyde dehydrogenase of Salmonella

enterica serovar Typhimurium LT2 and phaC1 gene of R. eutropha. A yield of

11.98% of cdw was achieved in two step fed batch fermentation using glycerol as

substrate (Andreessen et al., 2010).

2.7.5. Bacillus: Future PHA producer

A wide range of Bacillus spp. has been reported to accumulate PHB: B.

amyloliquefaciens DSM7 (11% of cdw), B. laterosporus (14%), B. licheniformis

(21%), B. macerans (28%), B. cereus (32%) and B. circulans (34%), B. firmus G2

(15%), B. subtilis K8 (32%), B. sphaericus X3 (36%), B. megaterium Y6 (48%), B.

coagulans (24%), B. brevis (41%), B. sphaericus ATCC 14577 (6%), B.

thuringiensis (52%), B. mycoides RLJ B-017 (69%) (Yilmaz et al., 2005; Porwal et

al., 2008; Singh et al., 2009). Bacillus sp. INT005 and B. cereus UW85 could

produce PHA with a wide range of compositions varying from PHB, P(3HB-co-

3HV), P(3HB-co-3HHx), P(3HB-co-4HB-co-3HHx) to P(3HB-co-6HHx-co-3HHx)

depending up on the substrate (Labuzek and Radecka, 2001; Tajima et al., 2003;

Valappil et al., 2008). Various Bacillus spp. have been shown by different

researchers to synthesize copolymers when co-fed with various substrates. Using B.

cereus UW85, the production of terpolymer of 3HB, 3HV and 6HHx was recorded

with ε- caprolactone as sole C source in mineral salts medium without any glucose.

However, addition of glucose along with ε-caprolactone seemed to suppress

copolymer synthesis and the result was the production of PHB (Labuzek and

Radecka, 2001). Bacillus sp. INT005 could accumulate PHB when glucose was used


32

alone as C substrate in the medium. However, addition of various C sources along

with very low glucose concentration resulted in copolymers of 3HB and 3HHx on

octanoate and decanoate, copolymers of 3HB-4HB-4HHx on 4-hydroxybutanoate

and 3HB-3HHx-6HHx on supplementation with ε-caprolactone (Tajima et al.,

2003). Recent studies have produced still more interesting information. B. cereus

SPV when grown on structurally unrelated C sources such as fructose, sucrose and

gluconate resulted in the incorporation of 4HB with the first two substrates and 4HB

and 3HV with gluconate in the medium (Valappil et al., 2007b). Although limitation

of nitrogen (N), phosphorous (P) and oxygen in the culture conditions are known to

influence PHB production, however, potassium limiting media led to the production

of a copolymer containing 3HB and 3HV monomers in contrast to only PHB under

sulphur, P or N limitation (Valappil et al., 2008). B. cereus CFR06 yielded PHB on

starch (Halami, 2008) and other Bacillus spp. could also produce PHB from

industrial food waste water, soya waste and malt waste from beer brewery plant and

PSS (Kumar et al., 2009). Bacillus sp. 256 utilize an unrefined natural substrate –

mahua (Madhuca sp.) flower as C source (containing 57% w/w as sugars) to

produce copolymers P(3HB-co-10% 3HV) (Anil-Kumar et al., 2007). Recently a

highy polymer yield of 61.07 % dcw, in 1L shaken flask study and 51.37 % dcw in

2L fermenter study has been reported, using B. cereus SPV with sugarcane molasses

as the main carbon source (Akaraonye et al., 2011). B. subtilis has been recently

used as a host for over expression of phaCAB genes from P. aeruginosa and R.

eutropha. Expression of phaC1 from P. aeruginosa and phaAB genes from R.

eutropha in B. subtilis resulted in the production of copolymers P(HD-co-HDD and

P(HB-co-HD-co-HDD) from malt waste (Wang et al., 2006). Since B. subtilis is not

a human pathogen, further supporting its usage as a host for expression of foreign

genes (Law et al., 2003). Here, B. megaterium phaPQRBC genes were cloned into

B. subtilis 1A304 (Φ105MU331). Recombinant B. subtilis could utilize malt waste

as a C source, further raising the hopes for producing PHA at cheaper rates. This

study showed that phaPQ of B. megaterium was essential for PHA production along

with phaRBC, although they could not observe any sequence homology in NCBI

database.


33

Several species of Bacillus, a Gram-positive bacterium have certain characteristics on

the basis of which they can be considered as potential candidate(s) for PHA production

on industrial scale. A brief description of their unique abilities is presented here.

(i) Lacks Lipopolysachharides (LPS)

Gram-negative bacteria such as C. necator (Vandamme and Coenye, 2004), A. latus

and recombinant E. coli are among those which have been exploited even for

industrial scale PHB production. However, in these organisms, the outer membrane

LPS are endotoxins (Stewart et al., 2006), which co-purify with the PHAs. Since LPS

induces a strong immunogenic reaction, this feature is undesirable for biomedical

applications of the PHAs (Chen and Wu, 2005; Valappil et al., 2007a). A review on

the toxic nature of LPS reveals that cyanobacterial (Gram- positive bacteria) LPS are

less toxic compared to those produced by members of Enterobacteraceae (Stewart et

al., 2006). Gram positive bacteria such as B. subtilis offers the advantage of lacking

LPS and excreting proteins at a high rate into the medium (Morimoto et al., 2008). It

thus stands a better choice as PHA producers for biomedical applications.

(ii) Possible candidate to replace E. coli as expression host

E. coli has been extensively used as a host for over expression of foreign genes,

including those involved in PHA biosynthesis. However, a major limitation of this

system is the secretion of proteins which result in the formation of inactive inclusion

bodies (Leonbartsberger, 2006; Chen et al., 2007). B. subtilis has been recently used

as a host for over expression of phaCAB genes from P. aeruginosa and R. eutropha. It

was chosen as host over E. coli, because of its short generation time, absence of

endotoxins and secretion of hydrolytic enzymes (amylases, proteinases, etc) for

utilizing biowastes as feed (Porwal et al., 2008), which may help in reducing the cost

of PHA production (Wang et al., 2006). Expression of phaC1 from P. aeruginosa and

phaAB genes from R. eutropha in B. subtilis resulted in the production of copolymers

P(HD-co-HDD) and P(HB-co-HD-co-HDD) from malt waste (Wang et al., 2006).

Since B. subtilis is not a human pathogen, it thus further supports its usage as a host

for expression of foreign genes (Law et al., 2003).


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(iii) Potential as hydrogen producer

The use of CO2 as a potential inexpensive renewable C source can help in reducing

PHA production cost (Tsuge, 2002). Synechococcus sp. MA19 was observed to

produce PHA upto 55% of cdw from CO2 (Nishioka et al., 2001). A. eutrophus was

shown to accumulate PHA at the rate of 1.55g/L/h, which was higher than that

recorded with cyanobacteria or photosynthetic bacteria (Tanaka et al., 1995). The

strategy being proposed is that if hydrogen (H2) production becomes cheap then R.

eutropha can produce PHB from CO2 and oxidation of H2, under dark fermentative

conditions (Ishizaki et al., 2001). Bacillus seems to meet the requirements of this

proposal. Bacillus spp. such as B. coagulans, B. licheniformis and B. subtilis have

been shown to evolve 1.5 to 2.36 mol H2/mol glucose (Kalia and Purohit, 2008).

Biowastes rich in starch such as damaged wheat grains have been employed as feed

for generating H2 (45 to 64 L/kg Total solids) by B. licheniformis and B. subtilis

(Sonakya et al., 2001). B. cereus strain EGU43 and B. thuringiensis strain EGU45

have been reported to generate 0.63 mol of H2/mol of glucose and up to 500 mg

PHB/L in different conditions (Porwal et al., 2008). More re cently, these strain have

been reported to produce both H2 followed by PHB on same media from glucose in

two stage system which further enhances the efficiency of the system (Patel et al.,

2011a).

2.7.6. PHA production by mixed microbial cultures (MMCs)

Among other factors causing high production cost is the sterilisation process. As an

alternative, using the combination of the MMCs on biowaste allows decreasing

operating costs by reducing the cost of substrate and that of the energy used (since no

sterilization is required) (Albuquerque et al., 2011). The use of MMC to produce

added value products (such as biochemicals and biomaterials) using ecological

selection principles to engineer the microbial consortium has been designated as

ecobiotechnology (Kleerebezem and van Loosdrecht, 2007). Enrichment of PHA

accumulating organisms is generally carried out by subjecting mixed cultures to

transient conditions of carbon supply, designated as Aerobic Dynamic Feeding or

Feast and Famine (Albuquerque et al., 2011).


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At early stages research was focused on the use of chemically defined media

supplemented with synthetic volatile fatty acids. Mixed microbial cultures collected

from activated sludge under feast and famine condition have been shown to

potentially producing PHA (Serafim et al., 2007; Jiang et al., 2008; Albuquerque et

al., 2010a, b). A maximum PHA content of 90% (PHA concentration; 4.3 g/L) from

lactate (Jiang et al., 2008) followed by 89% (PHA concentration; 1.2g PHA/g

substrate/h) from acetate (Johnson et al., 2009) and 78.5% from acetate pulse feeding

(Serafim et al., 2007) have been reported by mixed microbial cultures. However, the

use of low cost agro-industrial surplus feedstocks wastes and by-products is gaining

interest recently as reported by several authors e.g. fermented molasses (Albuquerque

et al., 2007; 2010a, b; Bengtsson et al., 2010a, b), fermented paper mill effluents

(Bengtsson et al., 2008), industrial wastewaters (Dionisi et al., 2006), fermented olive

oil mill effluents (Dionisi et al., 2005; Beccari et al., 2009), municipal wastewaters

(Coats et al., 2007), olive oil mill effluents (Dionisi et al., 2005; Beccari et al., 2009),

paper mill effluent (Bengtsson et al., 2008). Use of VFAs, which are the major

products of fermentation of biowaste, as feed for PHA production is the most

promising and efficient strategy employed by various researchers. Albuquerque et al.

developed a three-stage PHA production process from sugar cane molasses and have

reported a PHA content of 74.5% of cdw from VFA produced in fermented sugar

molasses attaining PHA concentration to the level of 0.49 g PHA/g substrate/h

(Albuquerque et al., 2007; Albuquerque et al. 2010a, b). An advantage with the use of

mixed culture is the production of co-polymers with a broad range of PHA

compositions. PHA containing different HA monomers can be produced on fermented

feedstocks (containing mixtures of organic acids such as acetate, propionate, butyrate

and valerate) without the need for added co-substrates as is the case for pure culture

fermentations and thus offers the possibility of producing PHA with a wide range of

thermal and mechanical properties. Lee et al., 2008 reported that pure culture

produced solely PHB and required large amounts of co-substrates (such as alcohols or

organic acids) to produce polymers with relatively small fractions of monomers (2-

8% HV) with same amount of feedstock. Co-polymers of P(HB-co-HV) with HV

ranging from 17 to 85% were reported (Lemos et al., 2006) with acetate and

propionate as substrate. 3HV content of upto 69 mol% is reported using mixed


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microbial cultures (Bengtsson et al., 2008; Arcos-Hernandez et al., 2010). Other

monomers reported include 3H2MV or 3HHx (Lemos et al., 2006; Bengtsson et al.,

2010b). A termopolymer of 3HB:3HV:3H2MV (molar ratio-6:58:24) was reported by

Lemos et al. (2006) from acetate/propionate mixtures.

Thus, mixed bacterial culture production has the potential to produce large amounts of

PHAs with seemingly lower costs. With the use of wastes and open cultures, a more

robust process could be obtained since, compared to pure cultures, mixed cultures are

more amenable to complex feedstocks. Although potential of mixed microbial culture

to produce PHA have been shown in early reports also, but potential of defined mixed

microbial cultures (consortia) to produce PHA have not been reported yet either on

synthetic media or biowaste.

2.8. Genomic status of PHA biosynthesis

The genes and enzymes involved in the biosynthesis of PHA vary in relation to their

organization among different organisms. The diversity of the PHB biosynthetic

pathways implies how far the pha loci have diverged. The phb (genes encoding

enzymes for SCL-PHA) and pha (genes encoding enzymes for MCL-PHA) are not

necessarily clustered and the gene organization varies from species to species (Reddy

et al., 2003). According to the need of organisms, with evolution phaC gene got

arranged with genes supplying monomers like phaAB and phaJ and while in some

organisms with genes involved in PHA regulation like phaZ and phaP (Naik et al.,

2008).

Among β-proteobacteria such as R. eutropha, Burkhelderia sp. A. latus, Delftia

acidovorans operon arrangement of PHA biosyhthesis genes is related to SCL-PHA

biosynthesis (class I PHA synthase gene) (Rehm, 2003). Here the genes are arranged

tandemly on the chromosome (Figure 2.6 & 2.7). P. acidophila, R. eutropha contains

the similar arrangement whereas Acinetobacter spp. contain the complete phaCAB

operon but genes are not in the same order (Rehm and Steinbuchel, 1999; Rehm,

2003; Suriyamongkol et al., 2007; Naik et al., 2008).


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Figure 2.6: Biosynthetic pathway and gene arrangement in Ralstonia eutropha

In bacteria belonging to α-proteobacteria such as Azorhizobium caulinodans,

Caulobacter crescentus, M. extorquens, Paracoccus denitrificans , Rhizobium metli

and Sinorhizobium meliloti containing class I PHA synthase genes phaC and phaAB

loci are unlinked or uninterrupted i.e. phaC is found elsewhere in the chromosome

(Rehm and Steinbuchel, 1999; Rehm, 2003). Other than α-proteobacteria, bacteria

such as such as Z. ramigera (β-proteobacterium), Aeromonas punctata (γ -

proteobacterium) and Gordonia rubripertinctus (a firmicute) contain similar

biosynthetic genes arrangement that are not co-localized (Naik et al., 2008).

Pseudomonas which produces MCL-PHA posses class II synthase e.g. P. oleovorans

and P. aeruginosa containing two different phaC genes i.e. phaC1 and phaC2

seperated by an additional phaZ gene encoding intracellular PHA depolymerase

enzyme (Figure 2.7). Enzymes encoded by phaC1 and phaC2 have similar primary

structures and substrate specificity. Downstream to the synthase gene arrangement,

phaD is located followed by phaI and phaF encoding structural and regulatory

proteins (Rehm, 2003). In bacteria having class III PHA syhthase, phaC and phaE

encoding the two subunits of polymerase enzyme are directly linked constituting a

single operon. phaEC and phaAB are present in one loci but are having opposite

orientation e.g. A. vinosum, Thiocystis violacea and T. pfennigii, Synechocystis sp.. In

Synechocystis sp. phaA and phaB do not map close to phaEC locus (Rehm, 2003). In

M. extorquens, Nocardia corallina, R. metli and Rhodococcus ruber an additional

gene phbP is present with an unknown function in the PHB regulation. Here also

phbCP and phbAB are present in the same locus but with different orientations


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therefore only the PHB polymerase encoding gene phbC has been identified so far

(Hustede and Steinbuchel, 1993). In A. caviae the PHB polymerase encoding gene

phbC has a flanking gene phbJ, which provides monomer for the enzyme. In C.

vinosum, P. acidophila, R. eutropha, R. meliloti and T. violacea an additional gene

phbF is present with an unknown function in PHA regulation (Suriyamongkol et al.,

2007). In P. acidophila, R. eutropha and Z. ramigera have a gene present upstream of

phbCAB operon and which is identical to the hypothetical E. coli protein YfiH (Naik

et al., 2008).

Figure 2.7: Genetic organization of representative PHA biosynthesis genes encoding the

various classes of enzymes (Rehm, 2003; Naik et al., 2008)

In Bacillus that contain class IV synthase phaR and phaC encoding PhaR and PhaC

subunits are present separated by phaB (McCool and Cannon, 2001; Rehm, 2003;

Valappil et al., 2007a). Screening of metabolic (KEGG) and genomic (NCBI) databases

for the presence of enzymes (Kalia et al., 2003) involved in PHA biosynthesis reveals that

genes for PhaA and PhaB are observed to be present in almost all the sequenced genomes

of Bacillus except certain strains of B. megaterium, B. thuringiensis, B. subtilis and


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Bacillus spp. (Table 2.5). However, quite a bit of variation is recorded in the case of phaC

gene and phaR. PhaC is frequently observed largely in the members of B. cereus group,

however, it's conserved domain is absent or partially present in most other Bacillus spp.

The presence of conserved domain of PhaR was largely partial or absent. In general B.

subtilis lacks genes related to PHA biosynthesis (Rehm, 2003) providing opportunity to

circumvent the need to eliminate or reduce the background effect caused due to

homologous genes of the host during heterologous gene expression. The concomitant

presence of PHA biosynthesis and depolymerization system has proved beneficial for

efficient production of PHA (Kleerebezem and Van Loosdrecht, 2007). Incidentally, B.

subtilis does contain phaZ encoding for PHA depolymerase.

Table 2.5: Conserved domains of enzymes for PHA biosynthesis and depolymerization in

Bacillus species

Organism PHA Biosynthetic enzymes β-Keto thiolase

Acetoacetyl reductase

Synthase Depolymerase

Conserved Domain PhaA PhaB PhaC PhaR PhaZ

Thiolase NADB FabG PhaC PRK 03918

DepA

Bacillus cereus Fa F F F Pb/Ac F B. thuringiensis F F F F P/A F B. anthracis F/NAd F F F P F B. coagulans F F F A A A B. coahuilensis F F F F A F B. weihenstephanensis F F F F A F B. megaterium NA F F F A F B. subtilis F/NA F/NA F/NA NA/A NA/A NA/A B. licheniformis F F F NA NA NA B. licheniformis F F F NA NA NA B. pumilus F F F A A A B. halodurans F F F A A A B. clausii F F F A A A B. selenitireducens F F F A A A B. amyloliquefaciens F NA NA NA NA NA

a: Full domain present b: Partial domain present c: Domain absent d: Not applicable


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As observed through this diverse arrangement of genes it can be concluded that

evolutionary forces has caused the clustering of phb genes in an operon with same

transcriptional units as in P. acidophila, R. eutropha, Acinetobacter sp., A. latus, A.

caviae and at times in separate transcriptional units as in Z. ramigera, P. denitrificans,

R. meliloli, C. vinosum, T. violaecae, P. oleovorans, P. putida etc. In some cases it

resulted in diverse orientation and varying flanking regions ultimately resulting in

diverged rearrangements in the phb loci.

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